1. Field of the Invention
The present invention relates to fragile X mental retardation 1 protein detection and, more specifically, to a system and method for quantifying the protein in tissue samples in a capture immunoassay using anti-FMRP antibodies.
2. Description of the Related Art
Fragile X syndrome (FXS) is a heritable condition characterized by cognitive and behavioral abnormalities and is the most common single gene cause of autism. The syndrome results from an expanded triplet CGG repeat that generates a fragile X site on the X chromosome that results in the absence or reduced expression of the FMR1 gene product FMRP. Almost all individuals with the syndrome carry FMR1 forms (alleles) that do not express the protein because the mutated alleles harbor long stretches of hypermethylated CGG repeats, e.g., the full mutation (FM) has more than 200 CGG repeats, that abolish or compromise FMRI transcription and/or translation. Alleles containing shorter repeats, e.g., permutations of 55 to 200 CGG repeats, may express a reduced amount of FMRP.
Most individuals carrying the permutation (PM) are not cognitively affected. However, PM alleles have been reported to play a role in autism spectrum disorders, premature ovarian failure and fragile X-associated tremor-ataxia syndrome. Early diagnosis of the syndrome is extremely important for child supportive care, early intervention and for family planning.
The laboratory diagnosis of Fragile X syndrome is currently performed by DNA testing (Southern blot and PCR methods) using blood or other tissues. The tests often require several days (7-10 days), however, and can be performed only by a limited number of specialized laboratories. These methods are directed at determining the length of the CGG repeats in the FMR1 alleles and use the CGG repeat size of the allele to infer or predict whether the proband cells produce abnormal levels of FMRP.
The development of an immunoassay for the direct quantification of FMRP has been hampered by the lack of mouse monoclonal antibodies (mAbs) having the affinity required to capture efficiently the human protein while showing no cross reactivity with the Fragile X related proteins, FXR1P and FXR2P. Various attempts have been made to test directly for the presence of FMRP in blood and other tissues using mAb IC3 that recognizes an epitope localized in the N-terminal of FMRP. The problem with these tests, however, is that the 1C3 mAb cross-reacts with the FX related protein FXRI and does not have a strong binding affinity to FMRP.
For example, one test uses a Western blot analysis to study FMRP expression in human lymphocytes from FM patients, PM and normal individuals. This test uses an anti-FMRP mAbla from Chemicon that cross reacts with a 70 kd protein (FXR1) in FM male samples. Immunocytochemical staining of lymphocytes or hair root cells using an anti-FMRP mAb (1C3) has also been used to detect cells expressing FMRP. Cells from male FM patients are not stained or show a small percentage of stained cells. The test does not measure the quantity of FMRP, but instead determines the fraction of cells that express the protein.
Finally, a luminometer-based sandwich ELISA for FMRP that allows quantification of FMRP in lymphocytes within 3 to 4 days has been described in the art. The assay uses a chicken polyclonal Ab to capture FMRP and a commercially available mAb, 1C3 (Chemicon, Millipore) for detection. Because of cross-reacting, this test produces a high background that does not allow signal detection when used with common blocking agents as milk and BSA. While the background may be reduced or suppressed using hydrolyzed casein as blocking agent, the blocking step—usually performed before incubation with the antigen—must follow the incubation with the antigen (lymphocyte extract) in order to produce a detectable signal and the ability of the chicken polyclonal Ab to capture FMRP from the lymphocyte extracts is reduced or abrogated by the presence of the blocking agent. In addition, this ELISA is cumbersome, time consuming (around 3 to 4 days), and requires several long incubation times, such as 24-48 hours for binding of the chicken Ab to the plate, overnight incubation for binding of antigen, about 2 hours for blocking, 8-10 hours for binding with the detecting mAb, and then about 12 hour for binding with horseradish peroxydase-conjugated donkey anti-mouse IgG. Accordingly, there remains a need for an immunoassay that is relatively fast and does not require a specialized laboratory.